Dietary fish oil modulates macrophage fatty acids and decreases arthritis susceptibility in mice.

B10.RIII and B10.G mice were transferred from a diet of laboratory rodent chow to a standard diet in which all the fat (5% by weight) was supplied as either fish oil (17% eicosapentaenoic acid [EPA], 12% docosahexaenoic acid [DHA], 0% arachidonic acid [AA], and 2% linoleic acid) or corn oil (0% EPA, 0% DHA, 0% AA, and 65% linoleic acid). The fatty acid composition of the macrophage phospholipids from mice on the chow diet was similar to that of mice on a corn oil diet. Mice fed the fish oil diet for only 1 wk showed substantial increases in macrophage phospholipid levels of the omega-3 fatty acids (of total fatty acid 4% was EPA, 10% docosapentaenoic acid [DPA], and 10% DHA), and decreases in omega-6 fatty acids (12% was AA, 2% docosatetraenoic acid [DTA], and 4% linoleic acid) compared to corn oil-fed mice (0% EPA, 0% DPA, 6% DHA, 20% AA, 9% DTA, and 8% linoleic acid). After 5 wk this difference between the fish oil-fed and corn oil-fed mice was even more pronounced. Further small changes occurred at 5-9 wk. We studied the prostaglandin (PG) and thromboxane (TX) profile of macrophages prepared from mice fed the two diets just before being immunized with collagen. Irrespective of diet, macrophages prepared from female mice and incubated for 24 h had significantly more PG and TX in the medium than similarly prepared macrophages from male mice. The increased percentage of EPA and decreased percentage of AA in the phospholipids of the macrophages prepared from the fish oil-fed mice was reflected in a reduction in the amount of PGE2 and PGI2 in the medium relative to identically incubated macrophages prepared from corn oil-fed mice. When this same fish oil diet was fed to B10.RIII mice for 26 d before immunization with type II collagen, the time of onset of arthritis was increased, and the incidence and severity of arthritis was reduced compared to arthritis induced in corn oil-fed mice. The females, especially those on the fish oil diet, tended to have less arthritis than the males. These alterations in the fatty acid pool available for PG and leukotriene synthesis suggest a pivotal role for the macrophage and PG in the immune and/or inflammatory response to type II collagen.

A sex-linked defect in the cross-linking of collagen and elastin associated with the mottled locus in mice.

A genetic abnormality in collagen and elastin cross-linking resembling experimental lathyrism has been identified in mice. The defect is an X-linked trait, attributed to the mottled locus which also influences coat color. The affected mice have aneurysms of the aorta and its branches, weak skin, and bone deformities in a spectrum of severity varying with the alleles at the mottled locus. A defect in the cross-linking of collagen was demonstrated in the skin of the affected animals by a marked increase in collagen extractability and a reduced proportion of cross-linked components in the extracted collagen. A decrease in lysine-derived aldehyde levels was found in both skin collagen and aortic elastin similar to that found in lathyritic tissue. Furthermore the in vitro formation of lysine-derived aldehyde was reduced. Thus the cause of the connective tissue abnormalities in these mice appears to be a defect in cross-link formation due to an impairment in aldehyde formation.

Functional alpha 1-protease inhibitor in the lower respiratory tract of cigarette smokers is not decreased.

Cigarette smoking is the major risk factor for the development of pulmonary emphysema, a disorder that may result from an imbalance between the elastase and antielastase levels in the lungs. Decreased functional alpha 1-protease inhibitor, an inhibitor of neutrophil elastase, might render smokers susceptible to elastase-catalyzed destruction of pulmonary elastic fibers and the development of emphysema. Binding and inactivation of isotopically labeled porcine pancreatic elastase and human neutrophil elastase by alpha 1-protease inhibitor were measured in fluid obtained by bronchoalveolar lavage of volunteers. The inhibition of elastase-catalyzed solubilization of elastin and a tripeptide substrate were also determined. The mean level of functional alpha 1-protease inhibitor in the bronchoalveolar lavage fluid of smokers was found to be equal to or greater than that of nonsmokers, contradicting reports by other investigators. Increased elastase derived from pulmonary neutrophils, rather than decreased functional alpha 1-protease inhibitor, appears to be the main factor in the genesis of emphysema in smokers.

The protein and lipid composition of arterial elastin and its relationship to lipid accumulation in the atherosclerotic plaque.

Elastin preparations from intimal layers and the media of normal and atherosclerotic human aortae were analyzed for protein and lipid content. In atherosclerotic aortae, elastin from plaques was compared with elastin from adjacent normal appearing areas of the same aorta. Arterial elastin purified by alkaline extraction appeared to be a protein-lipid complex containing free and ester cholesterol, phospholipids, and triglycerides. The lipid component of normal arterial elastin was small (1-2%). With increasing severity of atherosclerosis, there was a progressive accumulation of lipid in intimal elastin from plaques, reaching a mean lipid content of 37% in severe plaques. The increase in the lipid content of plaque elastic preparations was mainly due to large increases in cholesterol, over 80% of which was cholesteryl ester. This deposition of cholesterol in plaque elastin accounted for 20-34% of the total cholesterol content of the plaque. The increased lipid deposition in plaque elastin was associated with alterations in the amino acid composition of plaque elastin. In elastin from plaque intima, the following polar amino acids were increased significantly: aspartic acid, threonine, serine, glutamic acid, lysine, histidine, and arginine; whereas, cross-linking amino acids: desmosine, isodesmosine, and lysinonorleucine were decreased significantly. The amino acid and lipid composition of elastin from normal appearing aortic areas was comparable to that of normal arterial elastin except for intimal elastin directly adjacent to and medial elastin directly below the most severe plaques.The data indicate that the focal lipid deposition in early atherosclerotic plaques is due to a large extent to lipid accumulations in altered elastin protein of localized intimal areas. Continued lipid deposition in altered elastin appears to contribute substantially to the progressive lipid accumulation in the plaque. The study suggests that elastin of intimal elastic membranes may play an important role in the pathogenesis and progression of atherosclerosis.

Role of alpha-macroglobulin-elastase complexes in the pathogenesis of elastase-induced emphysema in hamsters.

Radiolabeled, enzymatically active or chloromethyl ketone-inactivated porcine pancreatic elastase was endotracheally instilled into hamsters. Gel filtration of the bronchopulmonary lavage fluid revealed two major radioactive fractions: one, eluting at 780,000 daltons, corresponding to an alpha-macroglobulin-pancreatic elastase complex, and another, at 68,000 daltons, corresponding to an alpha-1-protease inhibitor-pancreatic elastase complex. Elastolytic activity was recovered in the bronchopulmonary lavage fluid up to 4 d after elastase instillation and was associated with the alpha-macroglobulin-pancreatic elastase complex. Small amounts of this complex were recovered 14 d after instillation. When less than 1% (1.5--1.7 micrograms) of the usual dose of elastase was instilled into hamsters, the major radioactive complex was alpha-1-protease inhibitor-pancreatic elastase complex, and little or no elastolytic activity was found in the lavage fluid. In contrast to the instillation of 220 micrograms of elastase, no disease or hemorrhagic reaction was detected with this low dose, and without hemorrhage only insignificant amounts of alpha-macroglobulin-pancreatic elastase complexes were recovered from the lungs. To study the interaction of circulating antiproteases with elastase, hamster plasma was allowed to interact directly with the radiolabeled elastase; alpha-macroglobulin bound much more of the elastase than alpha-1-protease inhibitor, confirming the findings in the lung lavage experiments. The hamster's susceptibility to pancreatic elastase-induced emphysema may depend on the preferential binding of elastase to alpha-macroglobulin, which protects the elastolytic potential, rather than to alpha-1-protease inhibitor, which inactivates elastase. We speculate that if even a fraction of the residual radioactivity found in the hamster lungs as long as 144 d after instillation of elastase represents enzymatically active alpha-macroglobulin-pancreatic elastase complex, this could serve as a source of persistent elastolytic activity, which might explain the progressive nature of the pulmonary lesion.

Repair of protease-damaged elastin in neonatal rat aortic smooth muscle cell cultures.

The objective of this study was to investigate the elastin repair process in the rat aortic smooth muscle cell culture after proteolytic injury. Although little studied in vivo, elastin repair is thought to occur through a sequential process involving enzymatic removal (debridement) of damaged fibers followed by synthesis of tropoelastin, its subsequent processing, and eventual incorporation into new insoluble elastin. A second repair mechanism of proteolytically damaged elastin in a culture system is reported here. Repair in this system relates directly to restoration of resistance to elastin solubilization by hot alkali. As expected, severe injuries were observed with porcine pancreatic elastase (PPE). Using PPE, only 6% of the elastin, relative to control, was resistant to hot alkali immediately after elastase treatment. 4 wk later, resistance to hot alkali had increased dramatically to a mean of 90%. Repair took longer after injury with 75 micrograms of PPE as compared with 50 micrograms of PPE. The limited elastic fiber proteolysis induced by either human neutrophil elastase or porcine trypsin was repaired in culture within 2 wk. Elastin that had been radiolabeled with [3H]lysine 4-5 wk before injury was converted from a hot NaOH-susceptible to a NaOH-resistant elastin fraction during recovery from PPE injury. At the same time, the frayed elastic fibers that were seen with the electron microscope immediately after PPE treatment were replaced by continuous bands of elastin that resembled those in control cultures. Restoration of NaOH resistance did not require a net increase in total cell layer elastin, suggesting that relatively little new tropoelastin incorporation into the cell layer was required for this type of repair. These results suggested a salvage repair mechanism for proteolytically damaged elastin.

The appearance of free hydroxyproline as the major product of degradation of newly synthesized collagen in cell culture.

Embryonic lung fibroblasts and rabbit vascular smooth muscle cells have the ability to degrade newly synthesized collagen. Analysis of 24-h pulse media from cultures given [14C]proline demonstrates that greater than 90% of the degraded collagen is represented by free hydroxyproline rather than the peptide-bound imino acid. The addition of cycloheximide or alpha-alpha-dipyridyl to the culture medium during the pulse period severely diminished the formation of the free hydroxyproline demonstrating its enzymatic and protein (collagen) origin. It is proposed that assessment of free hydroxyproline formation may allow us to distinguish between intracellular and extracellular collagen degradation.

Lysine-derived cross-links in the egg shell membrane.

Egg shell membrane protein contains significant quantities of the lysine-derived aldehyde, allysine, and its aldol condensation product. NaB3H4 reduction followed by alkaline hydrolysis of purified protein revealed that there were six residues/1000 of both allysine and the reduced aldol while only traces of desmosine and isodesmosine were detected. The amino acid composition of the membrane protein did not resemble that of mammalian elastin.

Intrinsic enzyme activity associated with tropoelastin.

The presence of an enzyme(s) associated with purified tropoelastin has been established. Results indicate that the enzyme(s) remains closely associated with the soluble elastin throughout the entire purification procedure suggesting that it is very tightly bound. Enzymatic activity is optimum through the pH range 7-9 (37 degrees C) and can be inhibited by disodium ethylenediaminetetraacetate, N-ethylmaleimide, sulfite, soybean trypsin inhibitor and human alpha-1-antitrypsin. The fragmentation pattern appears to be specific and reproducible.

Incorporation of L-azetidine-2-carboxylic acid into hemoglobin S in sickle erythrocytes in vitro.

L-Azetidine-2-carboxylic acid, the naturally occurring lower homologue of L-proline, is incorporated into hemoglobin S (sickle hemoglobin) in vitro. Sickle erythrocytes from patients with sickle cell anemia incubated with L-[3H] azetidine-2-carboxylate synthesized radiolabeled hemoglobin which when isolated from cell lysates, co-chromatographed with hemoglobin S on DEAE-cellulose columns. The alpha/beta ratio of azetidine carboxylate incorporation into the globin chains of sickle hemoglobin was 0.94, which is consistent with the presence of four proline residues in each polypeptide chain. Incorporation of azetidine carboxylate into hot trichloroacetic acid-insoluble material in sickle erythrocytes indicated that the homologue was present in the polypeptide backbone of the globin chains of sickle hemoglobin. Amino acid analysis of the hot trichloroacetic acid-insoluble material from sickle erythrocytes which had been incubated with radiolabeled azetidine carboxylate indicated that 75% of the radioactivity could be accounted for as intact homologue while 20% of the radioactivity co-chromatographed with alanine. These results suggest that azetidine carboxylate is incorporated unaltered into hemoglobin S in addition to being metabolized to alanine in sickle erythrocytes prior to incorporation into protein. The kinetics of thermal precipitation of hemoglobin S (oxygen ligand) into which radioactive azetidine carboxylate or radioactive proline had been incorporated in vitro is identical. This observation, together with the behavior of hemoglobin S and the globin chains from hemoglobin S containing azetidine carboxylate during ion-exchange chromatography, indicates that homologue replacement of prolyl residues does not significantly alter the overall charge or stability of the hemoglobin S tetramer. Azetidine carboxylate did not inhibit uptake of radiolabeled proline by sickle erythrocytes suggesting that the homologue does not adversely affect amino acid transport in these cells.

Partial purification and characterization of a gelatin-specific protease from the culture media of human pulmonary alveolar macrophages.

A gelatin-specific protease from the culture media of human pulmonary alveolar macrophages has been partial purified by gel filtration and characterized. The macrophages were obtained by bronchopulmonary lavage from the lungs of disease-free smoking volunteers. The gelatin-specific protease initially requires trypsin activation. After chromatographing the culture media on a Sephadex G-200 column, trypsin is no longer required for activation. The gelatin-specific protease reported here shares many properties of previously reported gelatinases. It is inhibited by EDTA, cysteine, dithiothreitol and serum. It is unaffected by other protease inhibitors: phenylmethylsulfonyl fluoride, tosyllysine chloromethyl ketone and p-chloromercuribenzoate. Of all substrates tested activity was observed only with gelatin. It was inactive toward collagen, elastin and methemoglobin. This enzyme may have a role in the digestion of collagen which has been cleaved by a mammalian collagenase.

Isolation of purified insoluble aortic collagen.

The chemistry of collagen from major blood vessels, such as the aorta, is poorly defined because of problems encountered in solubilization techniques. Normal extraction of calf aorta with acetic acid and/or pepsin does not yield significant quantities of collagen. However, treatment of the aorta with purified pancreatic elastase results in a residue containing a significant portion of the collagen. The amino acid analysis, the acrylamide gel electrophoretic patterns and the electron micrographs of this residue display characteristics consistent with relatively pure collagen. Using this purified collagen preparation approximately 90% of the collagenous material present can be solubilized by pepsin treatment.

Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment.

The protein composition in the extracellular matrix of cultured neonatal rat aortic smooth muscle cells has been monitored over time in culture. The influence of ascorbate on insoluble elastin and collagen has been described. In the absence of ascorbate, the cells accumulate an insoluble elastin component which can account for as much as 50% of the total protein in the extracellular matrix. In the presence of ascorbate, the amount of insoluble collagen increases, while the insoluble elastin content is significantly less. When ascorbate conditions are varied at different times during the culture, the extracellular matrices are altered with respect to collagen and elastin ratios. The decrease in elastin accumulation in the presence of ascorbate may be explained by an overhydroxylation of tropoelastin. Approximately 1/3 of the prolyl residues in the soluble elastin fractions isolated from cultures grown in the presence of ascorbate are hydroxylated. Since the insoluble elastin accumulated in these cultures contain the unique lysine-derived cross-links in amounts comparable to aortic tissue, this culture system proves ideal for studying the influence of extracellular matrix elastin on cell growth and metabolism.

Effect of varying amounts of ascorbate on collagen, elastin and lysyl oxidase synthesis in aortic smooth muscle cell cultures.

In the presence of ascorbate, there is an increase in collagen synthesis with a concomitant decrease in insoluble elastin and lysyl oxidase activity in cultured rabbit aortic smooth muscle cells. While the addition of 0.5 micrograms ascorbate per ml of medium enhances collagen synthesis and accumulation, detectable insoluble elastin and lysyl oxidase activity remain essentially unchanged. However, at 2 micrograms ascorbate per ml, the integrity of the insoluble elastin is lost and lysyl oxidase activity is decreased. These studies suggest that by modifying the levels of ascorbate in the culture medium one can alter the nature of the extracellular matrix produced by the smooth muscle cells.

The synthesis of connective tissue protein in smooth muscle cells.

The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [14C] hydroxyproline when the cells were grown in the presence of [14C] proline. When the cells were pulsed with [14C] lysine, one could detect [14C] hydroxylysine and [14C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled epsilon-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [14C] lysine pulsed cells with NaBH4.

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli.

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a "sparing effect" on proline in the mutant. The incorporation of L-[U-14C]proline and L-[3H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologue was incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.

The interaction of collagen with alpha1-acid glycoprotein.

The influence of alpha1-acid glycoprotein on the formation of fibrous long spacing fibers of collagen has been investigated. It was observed that addition of the glycoprotein to dialyzed collagen solutions caused a significant decrease in the intensity of the circular dichroic spectrum of collagen. This phenomenon, which displays an optimum with respect to glycoprotein, is consistent with previous observations of fibrous long spacing fiber formation. Changes in viscosity of collagen initially dissolved in acetic acid were monitored during dialysis. It was found that a significant increase in viscosity must occur during dialysis of collagen before fibrous long spacing formation could take place. This increase in viscosity can be related directly to removal of acetic acid from the collagen solution. Removal of all sialyl residues from the alpha1-acid glycoprotein with neuraminidase prevents fibrous long spacing formation while removal of up to 35% of the sialyl residues has no effect on the interaction of glycoprotein with collagen. Amino acid composition and radioactivity studies suggest that 45-55% of the insoluble fibrous long spacing fibers is glycoprotein. In contrast to native collagen fibers, reduced fibrous long spacing fibers do not contain histidinohydroxymerodesmosine or hydroxylysinonorleucine. Instead, they contain significant quantities of allysine aldol and epsilon-hydroxynorleucine.

The structure of the carbohydrate units of the carboxyl-terminal peptide of procollagen as elucidated by 500 MHz 1H-NMR spectroscopy.

The oligosaccharides of chick embryo type I procollagen were isolated from carboxyl-terminal propeptide fragment by exhaustive digestion with papain and pronase, and then purified as a mixture of glycopeptides. The structures of the oligosaccharides were established by high-resolution 1H-NMR spectroscopy and found to be a mixture with respect to the non-reducing terminal residues as shown below: (formula; see text) The percentages refer to the relative amount of those mannose residues present in the mixture. The data suggest that the oligosaccharides are a microheterogeneous mixture of high-mannose type glycans containing between six and nine mannose residues per carbohydrate unit. Such carbohydrate chains, although not uncommon for glycoproteins, had never been found before for collagen or collagen-related compounds.

Plasma concentration of C-reactive protein and risk of ischemic stroke and transient ischemic attack: the Framingham study.

BACKGROUND: The role of C-reactive protein (CRP) as a novel plasma marker of atherothrombotic disease is currently under investigation. Previous studies have mostly related CRP to coronary heart disease, were often restricted to a case-control design, and failed to include pertinent risk factors to evaluate the joint and net effect of CRP on the outcome. We related plasma CRP levels to incidence of first ischemic stroke or transient ischemic attack (TIA) in the Framingham Study original cohort. METHODS: There were 591 men and 871 women free of stroke/TIA during their 1980 to 1982 clinic examinations, when their mean age was 69.7 years. CRP levels were measured by using an enzyme immunoassay on previously frozen serum samples. Analyses were based on sex-specific CRP quartiles. Risk ratios (RRs) were derived, and series of trend analyses were performed. RESULTS: During 12 to 14 years of follow-up, 196 ischemic strokes and TIAs occurred. Independent of age, men in the highest CRP quartile had 2 times the risk of ischemic stroke/TIA (RR=2.0, P=0.027), and women had almost 3 times the risk (RR=2.7, P=0.0003) compared with those in the lowest quartile. Assessment of the trend in risk across quartiles showed unadjusted risk increase for men (RR=1.347, P=0.0025) and women (RR=1.441, P=0.0001). After adjustment for smoking, total/HDL cholesterol, systolic blood pressure, and diabetes, the increase in risk across CRP quartiles remained statistically significant for both men (P=0.0365) and women (P=0.0084). CONCLUSIONS: Independent of other cardiovascular risk factors, elevated plasma CRP levels significantly predict the risk of future ischemic stroke and TIA in the elderly.

beta-VLDL-induced alterations in growth potentiating activity produced by mononuclear phagocytes.

This report describes the enhancement of growth potentiating activity produced by mononuclear phagocytes that were incubated with beta-migrating very low density lipoproteins (beta-VLDL). Conditioned media harvested from cultured human peripheral blood monocytes incubated in the presence or absence of the lipoprotein were evaluated for their ability to stimulate DNA synthesis ([3H]thymidine incorporation) of sparsely seeded quiescent BHK-21 (BHK) cells as well as neonatal rat aortic smooth muscle cells (NRSMC). Conditioned media from monocytes incubated in the presence of beta-VLDL enhanced [3H]thymidine incorporation into DNA of both BHK and NRSMC, when compared to conditioned media harvested from monocytes incubated in the absence of beta-VLDL. Studying NRSMC, this effect was evident using media collected from monocytes incubated with lipoprotein for 2 days; however, a longer incubation of monocytes plus lipoprotein was necessary to induce changes in growth potentiating activity for BHK cells. Likewise, the effect of beta-VLDL treatment of thioglycollate broth elicited BALB/c mouse peritoneal macrophages was evaluated. Conditioned media from lipoprotein-treated macrophages exhibited greater growth-stimulating activity for both BHK cells and NRSMC when compared to conditioned media from macrophages incubated in the absence of the lipoprotein. beta-VLDL did not affect viability of the mononuclear cells. These findings further implicate the involvement of the monocyte-derived foam cell in the development of atherosclerosis.